U.S. patent number 5,853,663 [Application Number 08/615,770] was granted by the patent office on 1998-12-29 for pressure distributor and multi-macrocarrier assembly for ballistic transfer transfection apparatus.
Invention is credited to Joseph Schroff, Matthias Schroff, Burghardt Wittig.
United States Patent |
5,853,663 |
Wittig , et al. |
December 29, 1998 |
Pressure distributor and multi-macrocarrier assembly for ballistic
transfer transfection apparatus
Abstract
The ballistic transfer transfection technology employs a cold
gas shock wave to accelerate microprojectiles that carry matter
into cells by mechanical force. The present invention relates to a
device that splits the cold gas shock wave into several individual
shock waves that spread the pressure of the initial shock wave to
several projectile launching devices. The number of cells
transfected by this new device is increased manyfold, compared to
the original apparatus. The use of the technology in a clinical
context is facilitated.
Inventors: |
Wittig; Burghardt (Lexington,
MA), Schroff; Matthias (Lexington, MA), Schroff;
Joseph (Lexington, MA) |
Family
ID: |
7757544 |
Appl.
No.: |
08/615,770 |
Filed: |
March 14, 1996 |
Foreign Application Priority Data
|
|
|
|
|
Mar 14, 1995 [DE] |
|
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195 10 696.2 |
|
Current U.S.
Class: |
422/50;
435/285.3; 435/52; 536/24.3; 536/23.1; 536/24.33; 89/1.14; 435/53;
514/44R |
Current CPC
Class: |
C12M
35/04 (20130101); C12M 35/00 (20130101); B65G
53/528 (20130101) |
Current International
Class: |
B65G
53/34 (20060101); B65G 53/52 (20060101); C12M
3/00 (20060101); G01N 001/14 (); G01N 015/06 ();
A61K 041/00 (); C07H 021/04 () |
Field of
Search: |
;514/44
;435/285.3,172.1,172.3,287,52,53 ;935/85 ;89/1.14 ;422/50,68 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Jones; W. Gary
Assistant Examiner: Rees; Dianne
Claims
What is claimed is:
1. An apparatus for the transfection of cells comprising:
a) a pressure separation means that divides a cold gas shock wave
into more than one pressure wave that propel macroprojectiles into
the direction of cells that are to be transfected by biological
matter,
b) a plurality of microprojectile launching devices and
c) a macroprojectile launching device holding means positioned
underneath said pressure separation means, holding in place said
plurality of macroprojectile launching devices so as to expose said
launching devices to the individual pressure bursts released by
said pressure separation means.
2. An apparatus according to claim 1, where the pressure separation
means diverts the cold gas shock wave into seven tubes generating a
partitioned cold gas shock wave wherein said seven tubes comprise
one central tube and six tubes arranged hexagonally around said
central tube, each tube connecting to one of seven macroprojectile
launching devices arranged in the same pattern as said seven tubes
and wherein said seven tubes are suspended directly underneath the
pressure separation means in a way which aligns each trajectory of
the partitioned cold gas shock wave with each one of said
macroprojectile launching devices.
3. An apparatus according to claim 2, wherein a pressure reducing
means is built into one or more of the tubes to adjust the pressure
exiting through said one or more tubes.
Description
FIELD OF THE INVENTION
This invention relates to an improved part of an apparatus used to
transfer matter into cells by accellerating particles in the
direction of the cells. The particles deliver any matter adsorbed
onto them upon passage through the cells.
BACKGROUND OF THE INVENTION
Many methods of modem cell biology require the transfer of matter,
mainly nucleic acids, into living cells (hereafter referred to as
transfection). Traditionally, this transfer of matter has been
important to both the fields of biological and medical research.
Recent progress, however, in the understanding of the body's
functions as regarded to molecular mechanisms has led to the idea
of treating human desease by using molecular approaches
(colloquially referred to as "gene therapy"). Many of the
biological methods suggested in this approach require the
transfection of somatic cells. A number of techniques have been
developed to achieve this aim: microinjection; electroporation;
transfection by viral vectors or liposomes; and direct bombardment
of cells with particles ("gene gun"). For a review on methods see
Methods in Enzymology 217, (1993), pp. 461-655, (Academic Press,
San Diego, Calif.).
Apart from microinjection, in which a single cell is injected
directly with the transfecting matter, these methods suffer from a
rather low and unreliable efficiency, efficiency being measured as
percentage of successfully transfected cells out of total of
treated cells. Microinjection's efficiency is very high; however,
the number of treated cells is generally too low for this technique
to be clinically valuable.
If the object of the transfection is to insert genetic information
into the cell, then successful transfection requires the passage of
the transfecting nucleic acid not only into the cell cytoplasm, but
into the nucleus. The nuclear membrane is a barrier more difficult
to cross than the cytoplasmic membrane. Many of the cells
transfected by means of electroporation or lipofection that have
incorporated the transfecting matter into their cytoplasm, will not
express any genetic message transferred into them. For expression
of any genetic message to happen, the genetic message has to pass
into the nucleus. The transfection of cells with DNA by
electroporation is most likely successful only when it happens
during cell division, because the division process momentarily
renders the nucleus permeable for the transfecting DNA.
In contrast, the ballistic transfection method achieves transport
into the nucleus by the kinetic energy of the passing particle. The
probability of nuclear passage of the microcarrier particle is
governed by the ratio of nucleus diameter to cell diameter, which
for many cells, is very favorable for nuclear passage. Thus, it can
be expected that any clinical approach to transfection of cells
with DNA would increase efficiency, employing the ballistic
transfection method.
A current estimate of the number of transfected cells needed in a
clinical protocol is in the order of 10.sup.7 -10.sup.8 cells. For
the reasons given above, we believe that of the transfection
methods mentioned, the ballistic transfer, i.e. directly bombarding
cells with particles that carry the transfecting matter into the
cells, has the greatest potential to achieve this aim.
Various embodiments of the idea of bombarding cells in order to
achieve transfection have been published. They differ in the
propulsion of the particles, the nature of the particles and
various other aspects. A number of patents have been filed
describing these embodiments (see: Jones, Frey, Gleason, Chee,
Slightom: Gas Driven Microprojectile Accelerator and Method of Use
U.S. Pat. No. 5,066,587; Jones, Frey, Gleason, Chee, Slightom: Gas
Driven Microprojectile Accelerator WO 9111526, U.S. Pat. No.
471,216; Sanford, Wolf, Allen: Apparatus for Delivering Substances
into Cells and Tissues in a Non-Lethal Manner EP 0 331 855; Tome:
Improved Particle Gun EP 0 397 413; Brill, McCabe, Yang:
Particle-Mediated Transformation of Animal Somatic Cells WO
91/00359; Mets: Aerosol Beam Injector WO 91/00915; WO 91/02071;
Johnston, Williams, Sanford, McElligott: Particle-Mediated
Transformation of Animal Tissue Cells WO 91/07487; Bruner, deVit,
Johnston, Sanford: Improved Method and Apparatus for Introducing
Biological Substances into Living Cells WO 91/18991; Bellhouse,
Sarphie: Ballistic Apparatus WO 9204439, GB 9018892.1). However,
only one embodiment to our knowledge, is commercially manufactured.
This embodiment is the "Biolistic" apparatus invented by John C
Sanford and manufactured under license from Cornell University and
DuPont by Bio-Rad (Hercules, Calif.). The propulsion of the
microcarriers is achieved in this embodiment by adsorbing the
microcarriers to a macrocarrier polymer sheet, which is accelerated
towards the cells by a cold gas shock wave. After retaining the
macrocarrier, the microcarrier sheaf continues towards the target
cell layer, eventually impacting and unloading the adsorbed
transfecting matter into the cells.
The method of ballistic transfection implies that only a (sometimes
large) fraction of the target cells is transfected successfully.
The microcarrier sheaf is rarely homogeneous, and has to be of
sufficiently small density in order not to kill too many of the
target cells, which invariably suffer from stress exerted on them
by both the shock wave and the impacting microprojectiles. A
balance must be found between a high survival rate and a high
transfection rate, which leaves part of the target cells
untransfected. For a new and successful procedure to separate
transfected cells from non-transfected cells, see our disclosure
"Method to Separate Cells that have been Modified by Ballistic
Transfer" (German application P 44 16 784.9).
As outlined above, the number of cells that need to be transfected
in any clinical use of "gene therapy" will probably be in the order
of 10.sup.7 -10.sup.8 cells, thus exceeding the efficiency of the
"Biolistic" apparatus commonly in use today. It would constitute a
great improvement to be able to increase tenfold the number of
cells transfected in one shot.
Within the concept of the "Biolistic" system, only a limited range
of improvements is feasible to achieve that aim. The number of
cells reached by the impacting particles is equal to the product of
area covered by the impacting microcarrier sheaf, by density of
cells in that area.
The area of cells on the petri dish that is covered by the
microcarrier sheaf depends on the distance between the macrocarrier
stop and the dish. However, this distance can not be increased
much, as a larger particle flight distance leads to a reduced
kinetic energy of the particles at the time of impact.
The size of the macrocarrier sheet can not be increased much, as
its mechanical properties are a consequence of its size.
The density of cells in a petri dish is at an optimum in a
monolayer, as several layers are not well penetrated by the
microcarriers, thus reducing transfection efficiency. In cell
culture, not many cell types easily grow more than one cell layer
in any case.
A possible solution is to increase the area covered in one
operation of the apparatus by accelerating more than one
macrocarrier, producing more than one microcarrier sheaf. The
present invention refers to such an arrangement.
BRIEF DESCRIPTION OF THE DRAWINGS
(All measures refer to millimeters, if not indicated otherwise)
FIG. 1 shows a view of the assembled headpiece and plate. For
clarity, only one macroprojectile launching device is shown.
FIG. 2 shows a section of the flange.
FIG. 3a shows a section, FIG. 3b a plan view of the pressure
distributor.
FIG. 4a shows a section of the insert that holds the retaining
grid, FIG. 4b shows a section of the ring plate in reverse
orientation.
FIG. 5a shows a section of the inserting plate and the plate that
receives the macroprojectile launching devices. The distance ring
is not shown. FIG. 5b shows a plan view of the same pieces.
FIG. 6 is an exploded view of inserting plate, distance ring and
receiving plate, and one macroprojectile launching device.
BRIEF SUMMARY OF THE INVENTION
The present disclosure relates to an improvement of the "Biolistic"
apparatus that enables a plurality of macrocarriers, and the
microcarriers adsorbed onto them, to be accelerated towards a
number of cells that is increased compared to the existing
apparatus.
According to the invention, the pressure entering the system and
propulsing the macrocarrier is divided into several tubes. The
tubes supply a plurality of macrocarrier launching devices with
fractions of the original pressure burst, leading to a plurality of
microprojectile sheafs impacting the target cells in an enlarged
area. The device holding the rupture disc, dividing the pressure
into several tubes supplying the macrocarrier launching devices
(hereafter referred to as "headpiece"), as well as the plate
holding the macrocarrier launching devices, said plate fitting into
the slots provided by the original apparatus, are object of the
present disclosure. The embodiment manufactured by us, consisting
of a headpiece supplying seven macrocarrier launching devices,
enabled us to transfect 1.times.10.sup.7 cells (erythroleukemia
cell line K 562) in one procedure, thus leading to a tenfold
increase of cell number.
DETAILED DESCRIPTION OF THE INVENTION
According to one preferred embodiment, the headpiece consists of a
flange 1 and a pressure distributor 2 with tubes 3 being fixed in
their position by a plate 4. The headpiece is manufactured as a
constructive unit and is not to be disassembled during the use of
the device. All parts are made of stainless steel and connected to
each other by industrial glue or welding. The flange 1 is equipped
with a thread corresponding to the thread in the "Biolistic"
apparatus manufactured by Bio-Rad, onto which it is screwed after
insertion of the rupture disc 5.
The plate 6 that receives the macroprojectile launching devices is
mounted onto the inserting plate 8 via the distance ring 7.
Optionally, a thread between 7 and 8 can be used to adjust the
relative vertical position of the macroprojectile launching
devices, which facilitates an adjustment of the particle
energy.
The plate 6 receives the inserts 9 that hold the grids 10 that
retain the macroprojectiles. On this lies the ring plate 11, which
receives the macroprojectile 12. The combination of insert 9 and
ring plate 11 is referred to as microprojectile launching
device.
EXAMPLE
The headpiece is screwed onto the "Biolistic PDS 100/He" apparatus
manufactured by Bio-Rad.RTM., Hercules, Calif. It is tightened with
the torque supplied by the manufacturer. The inserting plate 8 is
fit into the third groove from below. The plate receiving the
macroprojectile launching devices is turned in order to place each
of the launching devices directly under a tube ending. The plate
that supports the petri dish is fit into the lowest groove.
A suspension of colloidal gold (30 .mu.l, 1.6 .mu.m diameter, 30
mg/ml, Bio-Rad.RTM., Hercules, Calif.) is transferred onto each of
seven macrocarrier polymer sheets (Bio-Rad). The gold is allowed to
sediment, and the supernatant is removed. The gold is resuspended
in a mixture of one part aqueous solution of DNA
(fluorescein-endlabeled oligodesoxynucletides 50 .mu.g/ml) and one
part suspension of colloidal superparamagnetic particles (65 nm
diameter, Miltenyi GmbH, Bergisch Gladbach, Germany, used as
purchased). The suspension of superparamagnetic particles may be
dialysed against PBS (phosphate buffered saline) in order to remove
residual sodium azide added to the storage buffer. After
sedimentation, the supernatant is removed and the residual gold is
allowed to dry.
1.times.10.sup.7 -2.times.10.sup.7 cells (erythroleukemia cell line
K 562) in 10 ml RPMI medium (10% fetal calf serum) are transferred
onto a 9.8 cm petri dish and dispersed evenly. The cells are left
to incubate at a temperature of 37.degree. C. overnight (5%
CO.sub.2). The following day, supernatant medium is removed, the
cells are washed with ice-cold PBS, and all supernatant fluid is
removed carefully. This is very important, as any liquid covering
the cells decreases the transfection efficiency of the following
transfection dramatically.
The ballistic transfer is conducted according to the operating
protocol supplied by the manufacturer of the employed apparatus
(Biolistic PDS 1000/He, Bio-Rad). The rupture disc ruptures at 1550
psi. The pressure of the lower vacuum chamber is 508 mm (20 inches)
Hg.
The cells are resuspended after transfection in 1 ml ice cold
PBS/BSA medium (5 mM EDTA) and separated according to their
magnetic susceptibility, as described in our patent application
"Method to separate cells that have been modified by ballistic
transfer"(U.S. application Ser. No. 08/435,388; filing date May 5,
1995; European Publication No. 0686 697 A2, filing date May 8th,
1995):
The separation procedure is conducted on a MACS-separation column
(Miltenyi GmbH) according to the operating protocoll of the
manufacturer. The entire process is conducted at a temperature of
4.degree. C.: The cells are resuspended after transfection in 3 ml
ice cold PBS/BSA medium (5 mM EDTA) and washed onto the column
while in a magnetic field. The petri dish is washed again with 2 ml
PBS/BSA, which is added to the column. The column is washed with
three volumes of PBS/BSA medium (5 mM EDTA) at a flow rate of 0.3
ml/min. The fluid is retained and labeled N (non-magnetic). The
magnetic field is removed and the column is flushed with one volume
PBS/BSA medium (5 mM EDTA) in reverse direction to whirl up the
retained cells. The magnetic field is applied again and the fluid
is drained. The column is washed with four to five volumina of
PBS/BSA medium (5 mM EDTA) at a flow rate of 0.6 m/min.
The magnetic field is removed and the retained cells are washed
from the column by flushing with 3 ml of PBS/BSA medium (5 mM EDTA)
in short pulses. The collected fraction is labeled M (magnetic).
The collected fractions are subsequently assayed for their
fluorescence in a flow cytometry scanner (FACS) (Becton Dickinson,
Heidelberg, Germany).
* * * * *